CN111534444B - Method for high-density cultivation of Pichia pastoris fed-batch salting - Google Patents
Method for high-density cultivation of Pichia pastoris fed-batch salting Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 48
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 18
- 238000009938 salting Methods 0.000 title description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 102
- 238000000855 fermentation Methods 0.000 claims abstract description 81
- 230000004151 fermentation Effects 0.000 claims abstract description 81
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 63
- 150000003839 salts Chemical class 0.000 claims abstract description 53
- 239000000463 material Substances 0.000 claims abstract description 52
- 230000007797 corrosion Effects 0.000 claims abstract description 25
- 238000005260 corrosion Methods 0.000 claims abstract description 25
- 230000008569 process Effects 0.000 claims abstract description 20
- 230000001954 sterilising effect Effects 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 7
- 230000001502 supplementing effect Effects 0.000 claims abstract description 5
- 235000011187 glycerol Nutrition 0.000 claims description 33
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 12
- 238000005070 sampling Methods 0.000 claims description 11
- 238000012546 transfer Methods 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 6
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000013530 defoamer Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 6
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 6
- 235000011151 potassium sulphates Nutrition 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 239000013589 supplement Substances 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims 1
- 101150051118 PTM1 gene Proteins 0.000 abstract description 13
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000010790 dilution Methods 0.000 abstract description 2
- 239000012895 dilution Substances 0.000 abstract description 2
- 239000006052 feed supplement Substances 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 9
- 229910052751 metal Inorganic materials 0.000 description 6
- 239000002184 metal Substances 0.000 description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 3
- 238000003487 electrochemical reaction Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 150000002739 metals Chemical class 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000006012 monoammonium phosphate Substances 0.000 description 2
- 239000011684 sodium molybdate Substances 0.000 description 2
- 235000015393 sodium molybdate Nutrition 0.000 description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 2
- 238000011064 split stream procedure Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000000153 supplemental effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000011592 zinc chloride Substances 0.000 description 2
- 235000005074 zinc chloride Nutrition 0.000 description 2
- 229910000619 316 stainless steel Inorganic materials 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 229910000589 SAE 304 stainless steel Inorganic materials 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229910001567 cementite Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- QVCGXRQVUIKNGS-UHFFFAOYSA-L cobalt(2+);dichloride;hydrate Chemical compound O.Cl[Co]Cl QVCGXRQVUIKNGS-UHFFFAOYSA-L 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 238000006056 electrooxidation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 238000005111 flow chemistry technique Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- VXWSFRMTBJZULV-UHFFFAOYSA-H iron(3+) sulfate hydrate Chemical compound O.[Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O VXWSFRMTBJZULV-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- KSOKAHYVTMZFBJ-UHFFFAOYSA-N iron;methane Chemical compound C.[Fe].[Fe].[Fe] KSOKAHYVTMZFBJ-UHFFFAOYSA-N 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a method for culturing pichia pastoris by feeding salt in a high density, which comprises the following specific steps: firstly, putting a fermentation medium material into a fermentation tank, wherein the salt content in the fermentation medium material is 1/3 of the total salt content; secondly, sterilizing materials in the fermentation tank; thirdly, inoculating materials in the fermentation tank; fourthly, fermenting and culturing the material inoculated in the third step; fifthly, for the materials in the culture process in the fourth step, when the glycerol content consumption in the fermentation liquor in the fermentation tank is 0.1% that of the yeast, glycerol is supplemented, and salt is mixed in the supplemented glycerol; and sixthly, continuously supplementing methanol into the fifth step until the fermentation is finished. The beneficial effects of the invention are as follows: according to the characteristics of the properties and corrosion characteristics of the PTM1 salt, the PTM1 salt is respectively put into the culture substrate and the feed supplement raw material to play a role in dilution.
Description
Technical Field
The invention relates to the field of fermentation, in particular to a method for high-density culture of Pichia pastoris by adding salt.
Background
Pichia pastoris is a saccharomycete which can utilize methanol as a carbon source and an energy source, more target enzymes are required to be obtained in the culture process, PTMI salt is continuously added in the culture process, the method adopted at present is to add the PTM1 salt into a fermentation tank along with the methanol in proportion, and simultaneously, the PTM1 salt is fed into the fermentation tank to provide nutritive salt for fermenting the pichia pastoris, so that the healthy continuous growth of the yeast is ensured, in practical application, the PTM1 salt is corroded and leaked for less than one year on a 304 or 306 stainless steel pipeline, the methanol temporary storage tank and a methanol filter shell, potential safety hazards exist, the PTM1 salt needs to be replaced again, the cost is high, and safety accidents (residual flammable and explosive methanol) are very easy to occur in the process of replacement, and the PTM1 salt needs to be replaced by fire after a plurality of cleaning replacement inspection pass.
Disclosure of Invention
The invention overcomes the defects in the prior art and provides a method for culturing pichia pastoris by adding salt in a high-density manner.
The aim of the invention is achieved by the following technical scheme.
A method for culturing pichia pastoris by adding salt in a high density manner comprises the following specific steps:
firstly, putting a fermentation medium material into a fermentation tank, wherein the salt content in the fermentation medium material is 1/3 of the total salt content;
secondly, sterilizing the materials in the fermentation tank;
thirdly, inoculating materials in the fermentation tank;
fourthly, fermenting and culturing the material inoculated in the third step;
fifthly, for the materials in the culture process in the fourth step, when the glycerol content consumption in the fermentation liquor in the fermentation tank is consumed to 0.1% by yeast, glycerol is supplemented, salt is mixed in the supplemented glycerol, the salt amount is 2/3 of the total salt amount, and methanol is supplemented until the wet weight of the fermentation thalli reaches 180g/L, until the glycerol is supplemented;
and sixthly, continuously supplementing methanol into the fifth step until the fermentation is finished.
Further, the fermentation medium material in the first step is prepared by mixing the following components in parts by weight: 3-5 parts of glycerin, 0.11-0.13 part of potassium hydroxide, 3-5 parts of ammonium dihydrogen phosphate, 0.3-0.5 part of monopotassium phosphate, 1.5-1.7 parts of potassium sulfate and 0.014-0.016 part of defoamer.
Further, the fermentation medium material in the first step is prepared by mixing the following components in parts by weight: 4 parts of glycerin, 0.12 part of potassium hydroxide, 4 parts of ammonium dihydrogen phosphate, 0.4 part of potassium dihydrogen phosphate, 1.6 parts of potassium sulfate and 0.015 part of defoamer.
Further, the second sterilization method comprises the following steps: step one, heating materials in a fermentation tank to 121 ℃ by using steam, and maintaining the pressure for 30min; and step two, closing the steam and cooling to 30 ℃.
Further, the third step of seed transfer comprises the following steps: step one, sampling and evaluating a seed tank; step two, the seed transfer pipeline is sterilized by steam for 30 minutes and then transferred, and the inoculation amount is 10 percent.
Further, in the step one of the third seed transfer, the sampling time is once every 8 hours, and the detection content is PH detection, microscopic examination, wet weight, enzyme activity and coating of a detection plate.
Further, the conditions of the fourth culture step are as follows: the tank pressure is 0.03-0.05Mpa, the rotating speed is 120-135rpm, the temperature is 30 ℃, the air quantity is 1:1.5, and the PH value is 4.6-4.8.
Further, the amount of glycerol supplied per hour is controlled to be 30% as a quantitative index by controlling the oxygen solubility of the material in fermentation culture.
Further, the total amount of the supplementary glycerol in the fifth step is 2-3 times of the total amount of the fermentation medium material.
The beneficial effects of the invention are as follows:
the food fermentation liquid in the fermentation tank contains glycerol, and then the glycerol is supplemented, so that the problem of excessive glycerol addition in the fermentation tank for inhibiting growth is solved, and the secondary glycerol supplementation ensures the nutrition necessary for the wet weight growth;
according to the characteristics of the properties and corrosion characteristics of the PTM1 salt, the PTM1 salt is respectively put into the culture base material and the feed supplement raw material to play a role in dilution, meanwhile, the materials of the two tanks are continuously stirred, aggregation and electrochemical reaction can not occur, and corrosion can not occur after the process is improved and through one-section operation.
Detailed Description
The technical scheme of the invention is further described by specific examples.
Principle of galvanic corrosion: there are various corrosion principles of metals, of which electrochemical corrosion is one of the most widespread. When the metal is placed in an aqueous solution or in a humid atmosphere, the metal surface forms a microbattery, also called a corrosion cell (the electrodes of which are traditionally called cathode, anode, not called anode, cathode). Oxidation reaction occurs on anodeThe anode is dissolved, and the cathode undergoes a reduction reaction, generally acting to transfer electrons only. The reason for forming the corrosion battery is mainly that the metal surface absorbs moisture in the air to form a layer of water film, thus leading CO in the air to be formed 2 、SO 2 、NO 2 And the like are dissolved in the water film to form an electrolyte solution, and the metals immersed in the solution are always impure, such as industrial steel, and are actually alloys, i.e. contain graphite, cementite (Fe 3 C) As well as other metals and impurities, most of which are not iron-reactive. The anode of the corrosion battery thus formed is iron, and the cathode is impurity, and corrosion is continuously performed due to the close contact of iron and impurity.
And (3) phenomenon analysis: the PTM1 salt is corrosive to the 304, 316 stainless steel, the corrosion is stronger along with the increase of the concentration, the salt is aggregated under the static condition, the local concentration is more easily subjected to electrochemical reaction, and the electrochemical reaction is accelerated together with the methanol serving as an organic solvent.
1. Experimental data (laboratory 30L tank for experiment)
1. Experiment of influence of separate material flow salting process and original material flow salting process on fermentation result
(1) Salt adding process for material dividing flow
A method for culturing pichia pastoris by adding salt in a high density manner comprises the following specific steps:
firstly, putting a fermentation medium material into a fermentation tank, wherein the fermentation medium material is prepared by mixing the following components in parts by weight: 1.2 kg of glycerin, 0.036 kg of potassium hydroxide, 1.2 kg of monoammonium phosphate, 0.12 kg of monopotassium phosphate, 0.48 kg of potassium sulfate and 0.0045 kg of defoamer. 0.01% of PTMI salt (the composition of PTM1 salt can be 7 kg of ferric sulfate, zinc chloride, cupric sulfate pentahydrate, 1 kg of magnesium sulfate, 6 kg of cobalt chloride, 2 kg of sodium molybdate hydrate, potassium iodide, boric acid and concentrated sulfuric acid).
And a second step of sterilizing the materials in the fermentation tank, wherein the second step of sterilizing comprises the following steps: step one, heating materials in a fermentation tank to 121 ℃ by using steam, and maintaining the pressure for 30min; and step two, closing the steam and cooling to 30 ℃.
Thirdly, transplanting materials in the fermentation tank, wherein the third transplanting step comprises the following steps: step one, sampling and evaluating a seed tank; step two, sterilizing the seed transfer pipeline by steam for 30 minutes, and transferring seeds with 10 percent of inoculation amount; the first sampling time in the third seed transferring step is sampling every 8 hours, and the detection content is PH detection, microscopic examination, wet weight, enzyme activity and coating of a detection plate.
Fourthly, culturing the material after the seed transfer in the third step; the conditions of the fourth culture step are as follows: the tank pressure is 0.03-0.05Mpa, the rotating speed is 120-135rpm, the temperature is 30 ℃, the air quantity is 1:1.5, and the PH value is 4.6-4.8.
Fifthly, for the materials in the culture process in the fourth step, when the glycerol in the original fermentation liquid in the fermentation tank is consumed to 0.1 percent by yeast, adding salt into the glycerol to be supplemented, wherein the salt amount is 2/3 of the total salt amount in the original fed-batch methanol, (wherein the components of the fed-batch PTM1 salt can be 7 ferric sulfate hydrate, zinc chloride, cupric sulfate pentahydrate, 1 magnesium sulfate hydrate, 6 cobalt chloride hydrate, 2 sodium molybdate hydrate, potassium iodide, boric acid and concentrated sulfuric acid), and the methanol is supplemented simultaneously until the yeast thallus wet weight in the fermentation liquid reaches 180g/L, until the glycerol is supplemented; the amount of the glycerol supplied per hour is controlled to be 30% by taking the oxygen solubility of the material in the fermentation culture as a quantitative index, and the total amount of the glycerol supplied in the fifth step is 1/8-1/10 of the total amount of the material in the fermentation culture medium.
And sixthly, continuously supplementing methanol into the fifth step until the fermentation is finished, namely, the wet weight of thalli in the fermentation liquid is gradually increased, the activity of fermentation enzyme is not increased any more, the fermentation is finished, and the fermentation period is about 180 hours. The flow rate of the supplemental methanol is 3L/H/T-6.6L/H/T, based on the condition that the dissolved oxygen of the fermentation is not less than 30 percent.
(2) Original flow processing technology
The method comprises the following specific steps:
the first step, a fermentation medium material is put into a fermentation tank, wherein the fermentation medium material comprises the following components: 1.2 kg of glycerin, 0.036 kg of potassium hydroxide, 1.2 kg of monoammonium phosphate, 0.12 kg of monopotassium phosphate, 0.48 kg of potassium sulfate and 0.0045 kg of defoamer.
And a second step of sterilizing the materials in the fermentation tank, wherein the second step of sterilizing comprises the following steps: step one, heating materials in a fermentation tank to 121 ℃ by using steam, and maintaining the pressure for 30min; and step two, closing the steam and cooling to 30 ℃.
Thirdly, transplanting materials in the fermentation tank, wherein the third transplanting step comprises the following steps: step one, sampling and evaluating a seed tank; step two, sterilizing the seed transfer pipeline by steam for 30 minutes, and transferring seeds with 10 percent of inoculation amount; the first sampling time in the third seed transferring step is sampling every 8 hours, and the detection content is PH detection, microscopic examination, wet weight, enzyme activity and coating of a detection plate.
Fourthly, culturing the material after the seed transfer in the third step; the conditions of the fourth culture step are as follows: the tank pressure is 0.03-0.05Mpa, the rotating speed is 120-135rpm, the temperature is 30 ℃, the air quantity is 1:1.5, and the PH value is 4.6-4.8.
Fifthly, for the materials in the culture process in the fourth step, when the glycerol in the fermentation liquor in the fermentation tank is consumed to 0.1% by the yeast, starting to supplement glycerol, wherein the total amount of the glycerol is 3.6 kg of the volume of the fermentation liquor, starting to supplement methanol when the wet weight of fermentation thalli reaches 180g/L, and the amount of mixed salt in the methanol is 0.009 kg until the glycerol is completely supplemented; the amount of glycerol is supplemented every hour, and the oxygen solubility of materials in fermentation culture is controlled to be 30 percent as a quantitative index;
and sixthly, continuously supplementing methanol into the fifth step until the fermentation is finished, namely, the wet weight of thalli in the fermentation liquid is gradually increased, the activity of fermentation enzyme is not increased any more, the fermentation is finished, and the fermentation period is 180 hours. The flow rate of the supplemental methanol is 3L/H/T-6.6L/H/T, based on the condition that the dissolved oxygen of the fermentation is not less than 30 percent.
TABLE 1 experiment of influence of the split stream salt addition process and the raw stream addition process on fermentation results (grape oxidase fermentation 168H)
2. The experiment of the corrosion weight change of the equipment by the material-separating and salt-adding process and the original material-separating and salt-adding process is shown in table 2:
a small block of 304 steel pipe is placed in the methanol temporary storage tank, the weight is about 10 g, the weight is weighed (accurate to 1 mg) by using an electronic day before placing, the air-drying weighing record data is taken out every 30 days, and the corrosion rate calculation formula is as follows:
note that: w sample weight, w1 weight after corrosion, corrosion rate.
TABLE 2 weight variation of corrosion to equipment by split stream salt addition process and raw stream addition process
3. Data analysis
Through the comparative analysis of the two groups of data, the process of adding PTM1 salt into the separated material flow can completely replace the process of adding salt into the methanol flow, has no influence on the fermentation level, and solves the problem of corrosion of salt to equipment.
The foregoing describes two embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (9)
1. A method for preventing flowing salt corrosion equipment for high-density pichia pastoris culture is characterized by comprising the following steps: the method comprises the following specific steps:
firstly, putting a fermentation medium material into a fermentation tank, wherein the salt content in the fermentation medium material is 1/3 of the total salt content;
secondly, sterilizing the materials in the fermentation tank;
thirdly, inoculating materials in the fermentation tank;
fourthly, fermenting and culturing the material inoculated in the third step;
fifthly, for the materials in the culture process in the fourth step, when the glycerol content consumption in the fermentation liquor in the fermentation tank is consumed to 0.1% by yeast, starting to supplement glycerol, mixing salt in the supplemented glycerol, wherein the salt amount is 2/3 of the total salt amount, and starting to supplement methanol until the glycerol is completely supplemented when the wet weight of the fermentation thalli reaches 180 g/L;
a sixth step of continuously supplementing methanol into the fifth step until fermentation is finished;
the materials in the fermentation tank and the glycerol storage tank are continuously stirred.
2. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture according to claim 1, wherein the method comprises the following steps: the fermentation medium material in the first step is prepared by mixing the following components in parts by weight: 3-5 parts of glycerin, 0.11-0.13 part of potassium hydroxide, 3-5 parts of ammonium dihydrogen phosphate, 0.3-0.5 part of monopotassium phosphate, 1.5-1.7 parts of potassium sulfate and 0.014-0.016 part of defoamer.
3. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture according to claim 1, wherein the method comprises the following steps: the fermentation medium material in the first step is prepared by mixing the following components in parts by weight: 4 parts of glycerin, 0.12 part of potassium hydroxide, 4 parts of ammonium dihydrogen phosphate, 0.4 part of potassium dihydrogen phosphate, 1.6 parts of potassium sulfate and 0.015 part of defoamer.
4. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture according to claim 1, wherein the method comprises the following steps: the second sterilization method comprises the following steps: step one, heating materials in a fermentation tank to 121 ℃ by using steam, and maintaining the pressure for 30min; and step two, closing the steam and cooling to 30 ℃.
5. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture according to claim 1, wherein the method comprises the following steps: the third step of seed transfer comprises the following steps: step one, sampling and evaluating a seed tank; and step two, sterilizing the seed transfer pipeline by using steam for 30 minutes, and transferring seeds with an inoculum size of 10%.
6. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture according to claim 5, wherein the method comprises the following steps: the first sampling time in the third seed transferring step is sampling every 8 hours, and the detection content is PH detection, microscopic examination, wet weight, enzyme activity and coating of a detection plate.
7. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture according to claim 1, wherein the method comprises the following steps: the conditions of the fourth culture step are as follows: the tank pressure is 0.03-0.05Mpa, the rotating speed is 120-135rpm, the temperature is 30 ℃, the air quantity is 1:1.5, and the PH value is 4.6-4.8.
8. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture according to claim 1, wherein the method comprises the following steps: the amount of glycerol supplied per hour is controlled to be 30% as a quantitative index by controlling the oxygen solubility of materials in fermentation culture.
9. The method for preventing the flow salt corrosion equipment for high-density pichia pastoris culture of claim 8, wherein: and in the fifth step, the total amount of the supplementary glycerol is 2-3 times of the total amount of the fermentation medium material.
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| CN1699550A (en) * | 2005-05-17 | 2005-11-23 | 上海大学 | High density fermentation method for GS115/PFK-K5 |
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